ES2353032T3 - PROCEDURE FOR OBTAINING PROTEINS AND / OR VEGETABLE PEPTIDES, PROTEINS AND / OR PEPTIDES PRODUCED WITH THE SAME, AND USE OF THE SAME. - Google Patents

Abstract

Recovery of vegetable proteins and/or peptides comprises: (A) converting vegetable starting material to an aqueous matrix; (B) optionally separating off solid material and/or clarifying the matrix; (C) isolation of some or all of the proteins and/or peptides by adsorption on a polymeric ion exchange membrane; (D) optionally washing the membrane to remove impurities; (E) desorbing the proteins and/or peptides with an eluent; (F) isolating them from the eluent; and (G) optionally drying them. Independent claims are included for: (A) Proteins, peptides and/or mixtures of them obtained using the method; and (B) use of them in foods, animal feed and medicines.

Description

one

The present invention relates to a process for obtaining vegetable proteins and / or peptides, to proteins, peptides and mixtures thereof produced therewith, as well as to uses thereof.

For animal consumption both animal proteins are known I eat vegetables However, animal proteins, such as for example chicken proteins, milk proteins, such as casein or whey, they can present problems with regarding BSE, bird flu and other diseases. Proteins animals are also frequently related to the triggering allergies, even when these, as in In the case of a lactose intolerance, do not rely on the protein itself.

Vegetable proteins have problems with organisms genetically modified (GMO), its nutritional value and also with the triggering of allergies. The most vegetable protein known is soy protein. In the case of proteins vegetables also frequently presents the problem of flavor, for example in the case of soybeans, so that the food applicability is strongly limited. Until the the use of other proteins has not been imposed either vegetables, such as rapeseed, lupines or potatoes. In the case of rapeseed and legumes this could due, above all, to the fact that the fat content of these Starting materials generate rancidity.

From a chemical point of view, the percentage of protein of usual products on the market, which also contain many other desired and unwanted substances, consists of many individual protein and peptide molecules, which can subdivide first roughly so phenomenological in globulins and albumins. Globulins they have a spherical shape, therefore they are quite compact and

Water insoluble or at least sparingly soluble. The albumins have an open, more irregular shape and, by Therefore, they are soluble in water. In general, soluble proteins They are included under albumins. In addition, the usual protein in the market naturally consists of protein and peptide molecules They have different molecular weight. This does quite complicated handling, for example from the point of view of food technology, and can only be carried out a health assessment based on the spectrum of amino acids.

Consequently, the usual proteins in the market they have in common that they consist of a mixture of different protein and peptide molecules, and also contain components foreign to proteins, which come from the starting material original vegetable. To these belong for example glycosides, toxins (glycoalkaloids, trypsin inhibitors, etc.), antinutritive substances, such as acid phytic, which removes calcium and iron minerals from the digestion of humans and animals, since these are They eliminate and are not reabsorbed in the intestinal canal. Likewise fats and oils are included that are partly chemically bound to lipoproteins, as well as minerals.

So far, there are few pure proteins or peptides, for example for a healthy diet or as sales drugs free, that are cheap enough, for one more application wide. The reason is especially based on the use expensive treatment procedures, from industry Pharmaceutical An additional reason for the high price as well as an extremely limited availability is the origin of protein molecules, which are obtained from mammals

or from human secretions, for example blood serum, that all contain the desired action proteins in concentration reduced and are found available also only in limited quantity.

In documents US 2003113829, US 2003092151, US 2003092152, US 2003092150 and US 2003077265 is described by first time how they can be isolated in a relatively pure way

individual protein groups, from a mixture of proteins of a vegetable raw material, in this case of the potato.

These documents describe the choice of subject cousin; the method for the elimination of Kunitz inhibitors and of Bowman-Birk as well as of protein carboxyipeptidase Potato by heating, cooling, centrifugation and filtration of potato juice; the extension of the method by the use of an acid extraction agent together with a spraying of plant material in the solution of extraction, so that the protease inhibitor II is obtained; the method to control the performance and purity of protease II inhibitor during extraction by leaching of potato pieces, heating of the solution extraction, control of temperature, time and salt concentration, centrifugation and membrane filtration; isolation and purification of protease II inhibitors in A variant of the procedure.

However, in the case of known procedures it is disadvantageous that, although individual proteins are prepared or at less tightly defined protein groups, these procedures are however extremely complicated, Laborious and expensive. It must also be considered as a disadvantage A special selection of potatoes. This way I don't know limits only the availability of the raw material, but that through a necessary analytical control a stage is required Additional intermediate, complicated and expensive. In addition, logistics is complicated, since potatoes should be treated fresh and not They can be stored. Also, proteins can be damaged in the known procedures, since large ones are needed amounts of heat energy, since it works in diluted extraction solution and a high temperature for a long period of time, which It also requires large containers. Also in at a later stage additional energy is needed, since the amount of substance to be treated should be cooled to approximately room temperature for the other stages of

process. Organic acids are needed for extraction, that charge the environment of wastewater. In addition, the plant material shreds laboriously into particles

from about 100 hastam to 1,500 m. After extraction and additionally after heating, steps for the separation of solids are needed, to separate the coagulated or undissolved plant material from the protein solution and to be able to perform the last stage of ultrafiltration isolation. The known procedures finally provide only very few proteins, especially those that after a heating action at 100 ° C. over a period of approximately 3 hours have not yet been denatured. Finally, only those proteins that meet the aforementioned criteria, that is, protease inhibitor and carboxypeptidase inhibitors, are mainly obtained from the known procedures.

The invention is based on the objective of providing a procedure for obtaining proteins and / or peptides vegetables, which can overcome the disadvantages of state of the art A procedure is also provided, with which it is possible to obtain proteins and / or peptides vegetables on a broader basis of raw material, that is, It can be used not only to obtain potatoes, but from plants that contain proteins in general. Especially, the procedure may be performed in a manner harmless to the environment, with energy saving, simple and economical, and in this respect you will get any protein and peptide, pure or in mixtures, without limitations conditioned by the procedure.

Other objectives are to provide proteins and / or peptides produced according to the procedure as well as indicate Possibility of use.

These objectives are solved by a procedure according to claim 1, proteins, peptides or mixtures of the themselves according to claim 19 as well as possibilities of use

according to claim 20. Preferred embodiments are they follow from the respective dependent claims.

Surprisingly it was determined that the procedure according to the invention, unlike the procedures of the state of the technique can be carried out completely without a additional crushing of plants, heating stages and cooling as well as an extraction with organic additives. Especially the choice of proteins and proteins is not limited. Peptides to be obtained. The targeted choice of certain proteins and / or peptides can be achieved by controlling the selection procedure by precisely adjusting the procedural parameters As a result of the procedure they obtain either pure proteins without protein percentage strange or any extensive mixture of proteins that behave similarly in the adsorption process. By Therefore, protein purity can be adjusted at will according to the invention by the desorption step, for example in the form of dialysis. This can be advantageous especially when the quality of a previous product is sufficient to medical applications and only the final preparation should take place under sterile BPF conditions that the operator of the procedure given the case can not or wants to comply.

In other words, with the method according to the invention protein fractionation and / or can be achieved peptides of plant starting material in proteins or peptides individual or small groups of similar proteins with stages Extraordinarily smooth procedure, being able to still provide a large multitude of products and not being necessary any stage of expensive procedure or complicated

It has proved especially advantageous that according to invention fixation of the exchange groups takes place ionic on a membrane instead of on polymeric beads. The use of ion exchange membranes leads to a high flow rate, from none to few inlays, to an extraordinarily fast charge, since it is not needed no diffusion, reduced consumption of chemicals for

buffer solution and eluents, simple operation as well as a simple scale increase and the possibility of interconnecting anion and cation exchangers, since according to the Invention these are attached to different membranes.

Especially, with the method according to the invention it is possible to use only an ion exchange membrane, which it can be a cationic or anionic exchange membrane. Naturally, combinations of anionic and cationic exchange membranes. These can be in each case in any combination strong or weakly acidic

or alkaline It is conceivable that several can be connected cation exchange membranes and / or several membranes of anion exchange successively or in parallel. Likewise it is conceivable a serial connection of all the membranes of cation exchange and all exchange membranes anionic, however these two groups are connected Then in parallel. You can also conceive the mode of proceed in reverse, the membranes of cation exchange and anion exchange membranes in their respective groups in parallel, however connecting the Two groups in series. Therefore, all conceivable variations and are included within the scope of the invention.

Other advantages of the union of exchange groups Ionic to a membrane are as follows:

-A high load density allows packing in small volumes, which mean lower costs than, for example, in fixing on polymeric beads porous found in a bowl on a plate perforated and thus experience a flow to their around.

- No hair diffusion is needed or any diffusion of Fick so that the molecules that are going to adsorb themselves reach the ion exchange molecules, such as for example in the case of polymeric beads porous of synthetic or natural polymers. Only a convection takes place, since the dissolution

target flows directly over the membrane with the cargo carriers. In this way the procedure of Adsorption is clearly faster. In operation of circulation it is possible to pass the membranes several times and pores with load carriers, which accelerates clearly the adsorption procedure and also the desorption procedure Adsorption membranes can be used again or several times and are easy to clean.

-The membrane pore width can be adjusted to any between normal, micro, ultra and nanofiltration, according to the properties of the fluid and the substances to be treated and adsorbed. Therefore not a blockage or obstruction is possible as in the case of the pores of polymeric exchange resin beads ionic.

-There are the most diverse synthetic membrane materials in almost any choice, which allow extensive combinations of pressure procedure parameters (transmembrane), temperature and pH value.

-The type of construction of the modules in which they are made the membranes and that determine the type of technical construction of the membrane procedure can adapt to the procedure: plate modules, flow cross or rolled. The choice can take place among other things regarding the viscosity of the solid that It is retained in the membranes. If it is reduced, it is used preferably a module of the construction type rolled up, in which in a small volume you can incorporate a large membrane surface and therefore It is the most economical type of module construction.

-The module operation is possible in operation Batch or circulation. In the first case you can be introduced by pumping only so much dissolution target as permeate leave the facility. Whether Permeate flow ends, must be removed from the membrane retained, for example by washing. At

circulation operation the target solution circulates between the module and a storage vessel, so that the objective solution can be applied several times on the membrane. The solid contained in the retained is continuously removed from the container shows, so that in the modules governs a state stationary between two purification cycles. The procedure steps are described below.

of the method according to the invention with respect to a preferred embodiment, without wishing to limit without However, the object of the present application to it.

The process according to the invention for obtaining Vegetable proteins and / or peptides is described by Potatoes as plant starting material. Of those around

2,000 kinds of potatoes worldwide, approximately 50 classes are suitable for obtaining starch, since they contain a disproportionately large amount of starch, as a rule from 17 to 22% by weight. Instead, in principle all kinds of potatoes are suitable for obtaining proteins and peptides according to the process according to the invention.

After cleaning the potatoes, the first stage of procedure for obtaining starch is to be crushed Potatoes giving a fine pulp. From that pulp is then separate the potato juice (potato juice), which contains the protein and / or the peptide, of the solids, of the starch and of the fibers. The separation of starch and fibers can take place for example by centrifugation or hydrocyclones. The potato juice obtained contains approximately 20 g / l of potato protein, of which approximately 40% is patatin, a main reserve protein that belongs to glycoproteins, approximately 50% are inhibitors of Protease (IP) as well as 10% are high weight proteins molecular, to which the polyphenoloxidases, kinases and phosphorylase The patatin has a molecular weight of from 40 up to 44 kDa and consists of 363 amino acids. At a pH value of from 7 to 9 a dimer is formed with a size of from 80 up to 88 kDa. IPs are a heterogeneous class with seven

Subclasses of different proteins. Its function in the potato is the protein degradation, and therefore play a role important in the defense of the tuber against insects and pests microbial Protein degradation prevention is observed in the animal model as growth inhibition, it discusses an anticarcinogenic action and is promoted in part commercially the feeling of satiety of IP II. The classes Main IPs are IP I, IP II, cysteine inhibitor Potato protease (PCPI), Kunitz protease inhibitor (PKPI), carboxypeptidase (PCI), serine protease inhibitor (OSPI) and potato aspartyl protease inhibitor (PAPI).

Then a part of the proteins and / or are separated peptides by denaturation / coagulation, part that is not Desire or not necessary for precise separation. A denaturation / coagulation can take place for example by shifting the pH value, organic solvents or by precipitation with salts.

The potato juice obtained is clarified below in a microfiltration membrane device. In this regard the pore width of the membrane can be freely selected and It can be adapted to the desired products that will be obtained. Is possible a clarification of the potato juice obtained by example by centrifuges of all kinds of types of construction, provided a clear spin is obtained with exclusively dissolved components. These and all stages following, with the exception of drying in stage g), take place at a temperature below the coagulation temperature or denaturation temperature of proteins and / or peptides, preferably at a temperature below 30 ° C.

An essential part of the process according to the invention is the isolation of proteins and / or peptides from the aqueous matrix, in this case the potato juice, by adsorption on at least one ion exchange membrane, which It is composed of a synthetic polymer. Examples of such membranes can be purchased in the market with the name Sartobind® from Sartorius.

According to the invention it is possible that in step c) it is isolated only part of the proteins and / or peptides from the aqueous matrix by adsorption. This depends considerably from cation exchange membranes or Anionic used.

Similarly, directed desorption is of similar importance. of the proteins and / or peptides bound to the membrane of ion exchange through specially adapted eluents, after optional remnants of potato juice have been previously removed by membrane washing.

For the fixation of anionic proteins can be used ion exchange membranes with cationic groups, by example with trimethyl groups, while for proteins cationic must be present in the exchange membrane Ionic anionic groups, such as sulfomethyl groups.

For the mechanical protection of the adsorption membrane, but also to prolong its duration, it is recommended as previous stage a separation of solids and particles suspended or dispersed, as mentioned above. This can take place by a centrifuge or by a filtration, the latest in usual pore sizes of up to one microfiltration The use of a microfiltration of size

With an adequate pore of 0.2 µm, it has the advantage of letting all the proteins pass through, but simultaneously eliminating the microorganisms also contained in the protein-containing solution and therefore making the medium sterile. The proteins and / or peptides are then adsorbed on the membranes, the filtrate, permeate or clarified protein solution being pumped into the membrane adsorption module. With this, many procedure variants are possible. The cationic and anion exchange membrane modules can then be connected in parallel or in series. The adsorption membranes can be made in plate module, transverse or rolled flow construction modes. The feeding of the objective solution containing protein can take place in a terminal procedure

(Dead End) or in a circulation procedure. The first is necessarily a batch procedure, the latter can take place both in batches and continuously. The width of Adsorption membrane pore can be freely selected, but it is recommended that it is not smaller than the pores of the previous filtration, since otherwise the danger of accumulating material in the form of an adsorber retained that must be removed further then in the washing stage, and, since it consists of a potential product, It also means yield losses. If the membranes of adsorption are completely loaded with molecules of protein, which can be determined analytically so simple for example by measuring the conductivity in the effluent of the membranes or in the case of operation terminal in the permeate, the supply of the objective solution, and the membrane can be optionally washed To remove impurities. Washing can take place by water or a wash solution, but it should not denature proteins.

Products that adhere to membranes can desorber as in the case of a procedure of usual chromatography successively and selectively with several eluents This preferably occurs with a solution. saline, whose composition and concentration depends on the proteins and peptides to be eluted. Normally used sodium chloride and ammonium chloride solutions, without However, in this case the choice is almost unlimited and comes predetermined by the properties of proteins. It is also possible the addition of buffer salts or buffer solutions, by example phosphate buffer. So that eluted proteins are not denaturalize must be found in the eluent only in one reduced concentration Therefore, before drying it is advantageous A stage of concentration. In addition, by washing with water distilled or tap water can be adjusted at will purity of proteins isolated in this way. If you take for these two procedural steps an ultrafiltration membrane in a mode of construction of plate module, transverse flow

or rolled, in circulation operation, can be performed in this case the filtration and concentration simultaneously, by example continuously filling up as much water as washed in the sample as permeate leaves the pores of the ultrafiltration membrane. Purity control can be effectively controlled by measuring the electrical conductivity in the permeate.

In the next procedure stage a insulation of the product from the eluent, for example by drying or by separation of the eluent and the protein molecules on a pore width membrane suitable, which preferably extends in the range of from ultra or nanofiltration to reverse osmosis, they undergo diafiltration and concentration or just undergo concentration

or only to diafiltration.

As a last stage, drying optionally follows, being able to use a moderate lyophilization advantageously or a spray drying. Other types of drying, but an intense heat action that It could lead to damage to the product.

The following examples further illustrate the procedure according to the invention in greater detail.

Example 1

An anion exchange module with an area of 80 m2 with a binding capacity of 0.4 mg protein / cm2 can join 320 g of protein. 50% of the proteins in the juice of Potatoes are IP, that is approximately 10 g / l. After applying 32 Potato juice is then depleted capacity. This lasts to a typical flow rate of 300 l / h approximately 6.5 minutes The IP proteins can then be eluted.

Example 2

A cation exchange module with an area of 80 m2 with a binding capacity of 0.25 mg of protein / cm2 can join 200 g of protein. 40% of the proteins in the juice of Potato is patatina, that is approximately 8 g / l. Are needed

3.3 m2 of membrane for the complete attachment of the 1 l patatin of potato juice. Therefore, about 330 m2 can be absorbed 1 kg of potato juice of 125 l. Then you can elute the patatin

Example 3

A great advantage of the process according to the invention is the multiple use of both the membrane adsorber and the washing solution and eluents.

A cation exchange module is loaded with a surface of 15 cm2 with 1.5 ml a solution of ASB (ASB = bovine serum albumin) with a concentration of 10 mg / ml. This is somewhat below the maximum load of approximately 20 mg The scheme for the identification of the Long-term stability is realized by loading, washing, eluting and washing. The washing liquid is a potassium phosphate buffer 50 mM at pH 7, the eluent is a 1M NaCl solution in the same buffer A cycle lasts 21.5 minutes. Over time it they widen according to their nature the elution peaks, and are possible up to 65 cycles, without blocking the membrane and without break occurs. If the washing stage is prolonged 5 minutes more than 100 cycles are possible without cleaning the membrane. A blockage occurs from the 108th cycle. After cleaning with 0.5 M sodium hydroxide the membrane was free again, of so that you could start over with the procedure of production. In this way several thousand cycles are possible with an adsorption module, before it wears out.

Example 4

A disadvantage is the high consumption of water and salt in the washing and elution. Therefore, the multiple use of Solutions is very advantageous. The eluate is used after a cycle and a load with 0.4 mg of protein / ml again for the elution, and the load now amounted to 0.86 mg / ml. After the fourth elution concentration increased to 1.2 mg / ml. By consequently, the saving of eluent (water, salt and buffer) amounts to 75%.

The attached figure 1 shows a gel-based SDS-PAGE with the representation of total protein in potato juice before the treatment according to the invention as well as the proteins and protein fractions bound on membranes of cationic and anion exchange adsorption and eluted from again, which are obtained according to the procedure according to the invention.

As can be deduced from Figure 1, through from the anion exchange membrane, patatin could be isolated from directed way and through the exchange membrane cationic were able to isolate IP in a targeted manner and could separate in an essentially pure way, which highlights once again spectacularly the advantages of the procedure according to the invention.

The proteins and / or peptides obtained according to the invention they can be used for example in functional foods, it is that is, foods with positive physiological action. Can be used also for prevention and the fight against diseases as well as to improve performance and well-being. A use preferred of the proteins and peptides produced according to the invention could be in a pharmaceutical form, for example in capsule form In this case it is of special interest the protease II inhibitor, since its inhibitory effect is known of appetite and can easily be introduced for example in a hard gel capsule.

The features of the invention disclosed in the above description, in the claims as well as in the drawing can be essential both individually and in any combination for the implementation of the invention in its different embodiments.

Claims (16)

1. Procedure for obtaining proteins and / or peptides Vegetables comprising the stages: a) previously place a plant starting material which contains proteins and / or peptides in a matrix watery; b) optionally separate the solid components from the aqueous matrix and / or clarify the aqueous matrix; c) isolate at least part of the proteins and / or peptides from the aqueous matrix by adsorption on at least one exchange membrane ionic synthetic polymer; d) optionally wash the ion exchange membrane to remove impurities; e) desorbing proteins and / or peptides from the membrane of ion exchange with at least one eluent; f) isolate proteins and / or peptides from eluent; Y g) optionally drying the proteins and / or peptides isolated, in which the desorption of proteins and / or peptides individual or groups thereof in stage e) has place successively and selectively with the use of several different eluents and, before step c) precipitates and a part of the proteins and / or peptides is separated from of the aqueous matrix by denaturation / coagulation.

2.

Method according to claim 1, characterized because the aqueous matrix is obtained by crushing the material Starting vegetable to give a pulp or grinding materials Starting, especially dry, to give a flour e swelling in water and separating the solid components.

3.

Method according to claim 2, characterized because the solid components are starch and fibers of plant starting material.

Four.

Method according to one of the preceding claims, characterized in that the plant starting material is select from plants that contain proteins,

preferably potatoes, legumes, soy, rapeseed and mixtures of these, especially preferably potatoes.

5.

Method according to claim 4, characterized because legumes are selected from peas, beans, lupins, soybeans and mixtures thereof.

6.

Method according to one of the preceding claims, characterized in that the clarification in stage b) has place in a microfiltration membrane device.

7.

Method according to one of the preceding claims, characterized in that at least stages a) -f) are performed at a temperature below the temperature of coagulation or denaturation of proteins and / or peptides, preferably at a temperature below 30 ° C

8.

Method according to one of the preceding claims, characterized in that step c) and / or e) is carried or carried carried out in a batch or circulation procedure.

9.

Method according to one of the preceding claims, characterized in that in stage c) at least a cation exchange membrane and at least one anion exchange membrane.

10.

Method according to claim 9, characterized because the cation exchange membrane and the membrane anion exchange are operated in parallel or in Serie.

eleven.

Method according to one of the preceding claims, characterized in that each ion exchange membrane is found in an absorption module, preferably a plate module, cross flow or rolled.

12.

Method according to one of the preceding claims, characterized in that the pore width of the membrane of ion exchange is adjusted to obtain micro, ultra or nanofiltration

13.

Method according to one of the preceding claims, characterized in that the eluent is selected from a aqueous saline solution, preferably a solution of sodium chloride and ammonium chloride.

14.

Method according to one of the preceding claims, characterized in that the insulation in stage f) has place through membrane filtration or drying.

fifteen.

Method according to one of the preceding claims,

5 characterized in that drying in step g) takes place through spray drying or lyophilization. 16. Method according to one of the preceding claims, characterized in that denaturation / coagulation has place by shifting the pH value, solvents 10 organic or by precipitation with salts.